The epithelial-to-mesenchymal transition (EMT) is a cellular program in which epithelial cells gain mesenchymal characteristics and lose epithelial traits, which can result in increased motility and invasiveness. This process is necessary for proper development during embryogenesis, and for wound healing later in life. A similar aberrant, and more transient, program termed an oncogenic EMT can contribute to the metastatic dissemination of epithelial breast cancer cells. It is this metastatic spread of tumor cells that is often responsible for patient mortality. Using Twist1, a known EMT inducer, we recently discovered that microRNA- 424 (miR) is up-regulated during the active EMT process in HMLE human mammary cells. This occurs at a time when known mesenchymal markers are increasing, but epithelial markers have not yet been repressed. This temporal separation of events suggests there are different regulatory arms of epithelial and mesenchymal programming during an EMT, at least initially. As the majority of studies focus on the repression of epithelial genes, information is lacking on mesenchymal gene effects during an EMT. It is especially important to better examine this aspect of EMT, as invasive human breast carcinomas can simultaneously display mesenchymal- like and epithelial characteristics, setting the stage for the functional importance of pro-mesenchymal effects independent of loss of the epithelial phenotype. In line with this idea, our preliminary data indicate miR-424 is sufficient to induce th mesenchymal-like genes N-cadherin and vimentin, which typically correlate positively with breast cancer metastasis in vivo, without affecting epithelial programming. Additionally, miR-424 enriches for a tumor-initiation population in vitro. Thus, within this proposal we will operate under the hypothesis that miR-424 plays a role in promoting the mesenchymal-like programming arm of an EMT, contributing to increased stem cell-like features and breast cancer progression. We will determine if miR-424 is necessary for mesenchymal features downstream of Twist1 by inhibiting miR function, as well as examine the relevance of miR-424 to human disease by determining whether its expression correlates with increased aggressiveness in human breast cancers. In addition, we will examine whether miR-424 is involved in promoting tumor-initiation and breast cancer metastasis itself in vivo, as these properties have been associated with EMT. To perform these experiments, we will use cell lines that either over-express or inhibit miR-424 in both orthotopic and experimental metastasis mouse assays. Ultimately, this research plan will allow us to better understand the role of microRNAs in the mesenchymal arm of breast cancer-associated EMT, with the goal of identifying novel targets that can be inhibited to reduce breast cancer mortality.) PUBLIC HEALTH RELEVANCE: The spread of breast cancer cells to other sites in the human body, a process called metastasis, is the leading cause of death for breast cancer patients. Evidence suggests that metastasis is often fueled by the same processes that occur during normal development, and recently molecules called micro-RNAs, which are small naturally occurring regulators of gene expression, have been found to contribute to both normal development and tumor progression. By better understanding how these molecules operate out-of-context, researchers and physicians can develop new therapeutics to fight the spread of breast cancer.